1) Fibrosis Diseases and PDGF and PDGFR Family
When stimulated or damaged by outside factors, organ (or tissue) will form abnormal fibrosis, such as liver fibrosis, cardiac muscle, peritonaeum fibrosis, intervertebral fibrosis, bone marrow fibrosis, lung fibrosis, kidney fibrosis etc. Abnormal fibrosis could cause many diseases. For example, liver fibrosis causes liver cirrhosis, and liver cancer. Kidney fibrosis leads to malignant transformation of various renal tubule and tubulointerstitial Lesion, and is one of the major reasons for late-stage kidney failure. Lung fibrosis causes lung compliance decrease, lung capacity decrease, diffusion dysfunction decrease, VA/VQ imbalance, and finally lung failure, with lower than 50% of 5 year survival rate.
Research demonstrates that the cause of liver fibrosis, bone marrow fibrosis, lung fibrosis, and kidney fibrosis is related to the overexpression of PDGF family and PDGFR.
Three steps in chronic liver diseases are 1) hepatitis, 2) liver fibrosis, and 3) liver cirrhosis. Chronic fibrosis disrupts the essential structure of liver sinusoids, impairing the function of the liver and eventually leading to cirrhosis. If liver fibrosis cannot be treated, it will eventually lead to liver cirrhosis.
When liver cells die or affected by inflammation, Liver fibrosis forms by imbalance of extracellular matrix (ECM), with a bias toward deposition of fibrillar (type I) collagen within the subendothelial space of Disse. Viral hepatitis, chemical insult, and fatty liver all could cause liver fibrosis, a damaging liver disease. According to epidemic disease research reports, ¼ of hepatitis patients infected by virus will become chronic hepatitis patients. Among all chronic hepatitis patients, 5-20% will develop liver cirrhosis. Among liver cirrhosis patients, 50% of them will eventually develop liver cancer. Thus when liver fibrosis is formed, it is very hard to be reversed at normal condition. Liver fibrosis will lead to liver malfunction, which damages human health.
Although many different kinds of liver cells affect liver fibrosis, hepatic stellate cells (HSC) has essential function to the disease development.
In normal liver, the amount of HSC is quite small, with ratio of HSC:normal liver cells=1:20, and HSC's volume is 1.4% of the liver volume. HSC mainly has the following function: 1) store and metabolize Vitamin A; 2) Synthesize and secret small amount of ECM deposition, mainly Type I, III, and VI collagen; 3) break out to surround SEC (sinusoidal endothelial cells), support endothelial cells, and regulate sinusoid size; and 4) synthesize non-collagen glycoprotein and protein carbohydrate.
Research demonstrates that liver fibrosis is caused by activation of HSC in liver tissue. Over-expression of PDGF-C and PDGF receptors (PDGFR) at mRNA and protein levels is one of the earliest events. Activated HSC and myofibrobalsts produce a number of profibrotic cytokines and growth factors that perpetuate the fibrotic process through paracrine and autocrine effects. PDGF-BB and TGF-β1 are two key factors in fibrogenesis. Increased expression of both growth factors induces overexpression of collagen, and TIMP-1 and TIMP-2 secretion which inhibits collagen breakdown. All of these events lead to imbalance of ECM deposition and large deposition of collagen in liver tissue, which causes HSC to become myofibroblast-like (MFB). It leas to the surrounding liver cells wrapped by collagen, so that these liver cells lose normal function.
Liver fibrosis development leads to liver tissue hyperemia and liver steatosis formation, and eventually liver cancer. Thus any medication which can prevent or treat liver fibrosis is a good adjunct therapy for liver cancer.
Platelet-derived growth factor (PDGF) family has 4 types: PDGF-A, -B, -C, and -D), and two receptors: PDGFR-α and -β. They play an important role in wound healing, atherosclerosis, fibrosis, and malignancy. PDGF-C is the cytokine which was discovered most recently. It forms homodimer PDGF-CC, and has more potent biological activity towards mesenchymal cells than that of PDGF-AA, PDGF-AB, and PDGF-BB (Gilbertson et. al. JBC 276(29), 27406 (2001)).
Using real time PCR technology, Breitkopf team studied the mRNA expression profile of PDGF-C in transdifferentiating primary cultured HSC cells, an in vitro model system of hepatic fibrogenesis, either with or without stimulation of the cells with PDGF-BB or TGF-β1. When HSC cells transdifferentiating to MFB, PDGF-C mRNA was strongly induced: PDGF-C up to 5 fold from day 2 to day 8. This research demonstrates that PDGF-C may fulfill specific functions in hepatic fibrogenesis (Breitkopf et. al, Cytokine 31, 349 (2005)).
PDGF-C is a multi-domain protein (345 amino acids), consisting an N-terminal domain (residue 46-163), which is homologous to the CUB domain of neuropilin-1, NP-1, and a C-terminal domain (residue 235-245, GFD, growth factor domain), which has 23% homology with other PDGF members. These two domains can be separated by protease digestion in blood. PDGF-C can directly bind to PDGFR-α and PDGFR-β. According to competition assays and immunogenic precipitation experiments, PDGF-CC (PDGF-C homodimer) binds well with PDGFR-α and PDGFR-β. (Gilbertson et. al. JBC 276(29), 27406 (2001)). In addition, PDGF-CC strongly activate the tyrosine phosphorylation of PDGFR-α and PDGFR-β. GFD domain of PDGF-CC can bind well to PDGFR α/α or PDGFR β/β homodimer, and PDGFR α/β heterodimer.
Kidney fibrosis is the end result of pathological changes of renal tubes and intercellular substance. Kidney fibrosis is the general path for all kidney diseases and late stage kidney failure. The pathological characteristics are ECM deposition and loss of glomerular cells. The initiation and development of kidney disease are complex, which involve many factors, such as PDGF, TGF-β, and connective tissue growth factor (CTGF), and etc. PDGF affects kidney disease by inducing mesangial cell proliferation and renal tube and intercellular substance fibrosis. In normal rats, PDGF-AA is situated at renal papillary area, which affects cell migration; PDGF-BB is weakly expressed in renal tube and intercellular substance, which induces DNA synthesis and mitosis; PDGF-CC is expressed in glomerular endothelial cells, glomerular endothelial cells, vascular smooth muscle cells, glomerular capillary endothelial cells. When mesangial cells, kidney epithelial cells, and interstitial cells are damaged, PDGF-CC expression is increased. PSGF-CC's ability to activate mitosis is stronger than PDGF-AA, but weaker than PDGF-BB.
During chronic kidney inflammation process, stromal fibroblast cells proliferate, and its activated form is Myofibroblast (MyoF). MyoF can express α-smooth muscle actin (α-SMA), participating in tubulointerstitial fibrosis. Injecting PDGF-AA or PDGF-BB into mouse tubulointerstitial matter continuously for 7 days, PDGF-AA does not have any effect, but PDGF-BB induces tubulointerstitial fibrosis in a dose dependent manner. Li et al. discovered that PDGF-CC is the reason for fibroblast cells proliferation. Research data demonstrates that PDGF-CC abnormal expression is linked to tubulointerstitial fibrosis.
In anti-Thy1.1 nephritis mouse, PDGFR tyrosine kinase inhibitor significantly decreased mesangial cells proliferation, activated mesangial cells amount and Type IV collagen deposition. The mechanism is that STI571 inhibits ATP binding to Tyrosine kinase, so that phosphorylation and signal transduction are inhibited. The above data demonstrated that inhibitors to PDGFR pathway can significantly ease glomerulosclerosis.
Under pathological condition, PDGF expression increases. Overexpressed PDGF causes tubulointerstitial renal cell transfomation, inflammatory cell infiltration, and cytokine production, which leads to tubulointerstitial fibrosis and malignant renal diseases. Decrease or inhibition of PDGF synthesis or activity has certain effect on treating kidney fibrosis.
PDGF-B or PDGF-D injection or overexpression induces vascular mesangial proliferation and kidney fibrosis. Interference experiment demonstrated that PDGF-C induces tubulointerstitial fibrosis. PDGF-B and -D are key factors for vascular mesangial proliferation and kidney fibrosis. Thus PDGF family is growth factor for kidney diseases, and strongly stimulate the proliferation of mesangial cells.
Currently, this field is in need of an effective product to prevent and treat fibrosis diseases, such as liver fibrosis, kidney fibrosis, and lung fibrosis.
2) Cancer
Dimerization and autophosphorylation of PDGFR occur upon receptor-ligand interaction. Phosphorylated tyrosine residues, in the context of specific amino acid residues, interact with Src homology 2 domains (SH2) of intracellular signaling molecules. These include phospholipase γ (PLC-γ), Ras GTPase-activating protein (Ras-GAP), p85 subunit of PI3K, growth factor receptor-bound protein 2 (Grb 2), Syp (tyrosine-specific phosphatase), Src homology and collagen protein (Shc), and Src. These signaling molecules further transduce signal transduction pathways by activating downstream signaling molecules such as mitogen activated protein kinase family members (ERKs, JNKs), and focal adhesion kinase (FAK, a mediator of integrin signaling pathway) among others. These signals enter the nucleus and stimulate expression of a set of immediate-early-response genes that mediate PDGF-induced cellular processes including cell cycle, cell migration, and transformation.
Studies during the past two decades clearly indicate the significance of PDGF in human tumors. In vitro, overexpression of the v-sis oncogene product (p28v-sis) or PDGF-B in cells that express these receptors enhances transformation, indicating autocrine mechanism in tumorigenesis. Recent studies revealed a critical role for paracrine PDGF signaling in carcinogenesis through the regulation of epithelial-stromal interactions. Using nude mice, it was demonstrated that PDGF activation of stromal cells results in tumorigenic conversion of immortal human keratinocytes. Enhanced PDGF immunostaining was detected in soft tissue tumors and advanced breast tumors.
The fatal phenotypes of PDGF- or PDGFR-deficient mice include cardiovascular and hematological defects. PDGFs produced by endothelial cells in vessels promote recruitment and proliferation of vascular smooth muscle cells/pericyte progenitors expressing PDGFR. Chemotactic and mitogenic activities mediated by the PDGF/PDGFR paracrine signaling loop are crucial for the formation, branching, and maintenance of blood vessels. As in embryogenesis, PDGF plays a critical role for angiogenesis in human tumors. Tumor angiogenesis, required for tumor outgrowth and metastasis, is a complex and highly regulated process involving many different cell types and extracellular factors. PDGF is also involved in angiogenesis and tumor metastasis. Thus inhibiting PDGF signaling pathway could potentially attenuate primary and metastatic. (Yu et al., J of Biochem and Mol Bio, 36(1), 49 (2003)).